1. Field of the Invention
The present invention relates to a monoclonal antibody that binds to a single-chain type IV collagen polypeptide secreted as a single-chain form of a type IV collagen gene product and suppresses the growth of tumor tissues expressing the single-chain type IV collagen polypeptide. The present invention also relates to a pharmaceutical drug and a diagnostic, preventive, or therapeutic drug for tumor containing the monoclonal antibody.
2. Description of the Related Art
Living tissues are composed of cells, which are units of life, and extracellular matrices present around or between the cells. The extracellular matrices are large protein complexes composed mainly of sugar-modified proteins such as collagen, laminin, elastin and proteoglycan. The collagen accounts for approximately 30% of biological proteins and is abundantly found in connective tissues. Also, the collagen constitutes basement membranes at the boundary between epithelial and connective tissues or at the boundary between endothelial and connective tissues. The functions of the extracellular matrices are known to be involved in the control of cell growth and/or differentiation. The interaction between the extracellular matrices and cells has been shown to be important for the development, repair, regeneration, or the like of normal tissues.
This interaction between the extracellular matrices and cells has also been shown to play an important role in tumor cell growth and angiogenesis during tumor formation. Tumor cells reconstruct an extracellular microenvironment by the repetitive degradation and remodeling of the extracellular matrices. As a result, the growth of the tumor cells or the formation of new blood vessels is promoted to allow tumor tissues to grow. Also, the invasion and/or metastasis of the tumor cells are known to occur due to the degradation of such remodeled extracellular matrices.
The collagen is known to include 20 or more types differing in genotype. For example, mainly type I collagen is present in connective tissues composed of fibroblasts, interstitial cells, and the like, while mainly type IV collagen is present in basement membranes.
The type IV collagen is secreted from various cells such as epithelial cells and endothelial cells, in addition to fibroblasts or interstitial cells. The type IV collagen molecules thus secreted are considered to associate with one another to construct the basement membrane skeleton having a mesh structure. The basement membranes of normal tissues are present at the boundary between epithelial and interstitial tissues, the boundary between endothelial and interstitial tissues, or the like and regulate tissue morphology and functions.
The collagen molecule has a loose right-handed triple helix structure composed of three polypeptide chains together, each of which has the conformation of a left-handed helix. The type IV collagen includes 6 genotypes. α1 to α6 polypeptide chains derived from these 6 genes are known and thought to be variously combined to form three or more types of molecular species. The most widely found basement membranes are constituted of aggregates of the type IV collagen molecules each having two α1 polypeptide chains and one α2 polypeptide chain cross-linked through intermolecular disulfide bonds. By contrast, the type IV collagen molecules composed of α3 to α6 polypeptide chains are found only in the basement membranes of a limited number of tissues.
As described above, the type IV collagen refers to a trimer molecule having a triple helix structure formed by three a polypeptide chains cross-linked through intermolecular disulfide bonds, or an aggregate of these trimer molecules. In general, the type IV collagen is known to be extracellularly secreted as trimer molecules to form aggregates outside the cell. However, the present inventors have found that cultured human cells secrete the type IV collagen as well as its single polypeptide chain (hereinafter, also referred to as a “single-chain type IV collagen polypeptide”) that neither has an intermolecular disulfide bond nor takes a helix structure (see Takahashi, S. et al., Connective Tissue (1999) vol. 31; pp. 161-168). Particularly, cells cultured in a vitamin C-free medium produce the single-chain type IV collagen polypeptide in a much larger amount than that of the type IV collagen (see Yoshikawa, K. et al., J. Biochem. (2001) vol. 129; pp. 929-936). Also, the type IV collagen is known to undergo degradation by a protein-degrading enzyme (gelatinase, matrix metalloprotease-2, etc.). Because of lacking a helix structure, the single-chain type IV collagen polypeptide is more susceptible to degradation by a degrading enzyme than the type IV collagen having a triple helix structure. Accordingly, it has been assumed that the full-length single-chain type IV collagen polypeptide is difficult to detect in vivo. Nevertheless, the present inventors have found the single-chain type IV collagen polypeptide in human placenta (see Kajimura, D. et al., Biochemical and Biophysical Research Communications (2004) vol. 314; pp. 11-16).
The present inventors have further revealed that the single-chain type IV collagen polypeptide secreted from cells undergoes a posttranslational modification different from that of the type IV collagen. Proline hydroxylation and lysine hydroxylation in the type IV collagen are known to participate in the formation and/or stabilization of the triple helix structure, whereas the degrees of proline hydroxylation and lysine hydroxylation in the single-chain type IV collagen polypeptide are lower compared with the type IV collagen. In addition, the single-chain type IV collagen polypeptide reacts with Agaricus bisporus agglutinin (ABA) lectin that recognizes a sugar chain Galβ1-3GalNAc, whereas the type IV collagen does not react with ABA lectin.
As described above, the single-chain type IV collagen polypeptide has a chemical structure different from that of the type IV collagen or a polypeptide resulting from the denaturation thereof. The single-chain type IV collagen polypeptide is thus presumed to have biological functions different from those of the type IV collagen.
Solid tumor requires new blood vessels for its growth. For this reason, the inhibition of angiogenesis is one of methods for inhibiting tumor growth. The type IV collagen has collagenous and non-collagenous regions. Arresten, a polypeptide contained in the C-terminal non-collagenous region, reportedly inhibits lumen formation and inhibits angiogenesis, resulting in the suppression of tumor growth (see Thomas M. Mundel et al., Microvascular Research (2007) vol. 74; pp. 85-89).
JK199 (see Japanese Patent Application Laid-Open (JP-A) No. 63-78067) and antibodies contained in assay kits for the type IV collagen in serum (see Instruction manual of the type IV collagen assay kit “Panassay IV-C Latex”; Daiichi Pure Chemicals Co., Ltd. (2006 revised edition); Obata, K. et al., Clinica Chimica Acta 181, pp. 293-304, 1989; and JP-A No. 2-1553) have been reported as monoclonal antibodies against the type IV collagen.
In addition, an antibody acting on a “cryptic collagen site” (see JP-A No. 2009-240324) has been reported as a monoclonal antibody against denatured type IV collagen. However, it has been neither disclosed nor suggested that these antibodies recognize the single-chain type IV collagen polypeptide that is extracellularly secreted and stably present.
Meanwhile, JK132 (see Takahashi, S. et al., Connective Tissue (1999) vol. 31; pp. 161-168 and Iwata, M. et al., Journal of Biochemistry (1995) vol. 117 (6); pp. 1298-1304) has been reported to recognize the single-chain type IV collagen polypeptide, as an antibody recognizing the single-chain type IV collagen polypeptide. Unfortunately, these reports have not revealed the biological role of the single-chain type IV collagen polypeptide or its relation to cancer. Thus, it has been neither disclosed nor suggested that a pharmaceutical drug containing an antibody against the single-chain type IV collagen polypeptide is useful as diagnostic and therapeutic drugs for tumor.